Bacterial polysaccharides are excellent targets for vaccine development as they are surface exposed and often the most protective antigens expressed on a pathogen. However, purified polysaccharides are generally poor immunogens, particularly if their immunogenicity is not enhanced by conjugation to a protein. Such polysaccharide-protein conjugate vaccines are often complicated and expensive to produce and difficult to deliver. In order to demonstrate the potential of a novel approach to utilize polysaccharides as vaccine candidates, we have expressed the O antigen portion of Pseudomonas aeruginosa lipopolysaccharide (IPS) on an attenuated strain of Salmonella typhimurium. We compared different routes of immunization to protect against acute P. aeruginosa pneumonia in a mouse model system and found that intranasal administration of the vaccine provides better protection than either oral or intraperitoneal delivery. However, we do not know whether it is the location, level, and/or type of antibody response that is responsible for this protection. We have also shown that intranasal delivery of this vaccine can protect mice against P. aeruginosa infections after corneal trauma or after burns, but do not know the optimal route of immunization to induce immunity at these different sites, nor do we know the effect of host factors such as cystic fibrosis on modulating the immunity induced by the attenuated recombinant S. typhimurium. Also unclear is whether such vaccines can alleviate problems associated with polysaccharide-protein conjugate vaccines including subtype specificity and subtype inhibition, and the exclusion of acid-liable O antigens. The specific aims of this grant are to (1) determine the basis for protective immunity to P. aeruginosa acute pneumonia mediated by the attenuated recombinant S. typhimurium O antigen-based vaccine, (2) determine the optimal route of immunization to protect against P. aeruginosa infections after corneal injury or burns, (3) determine whether the vaccine can protect CF mice from P. aeruginosa lung infections, and (4) test additional O antigen-based vaccines for polysaccharide expression, immunogenicity, and protection. Overall, the long-term goal of this project is to understand the basis of protection against these varied infections, which in turn will allow us to develop rational strategies for vaccine development against this important opportunistic pathogen. We anticipate that this knowledge will also be applicable to other pathogens.